Compounds and methods of use to derivatize neighboring lysine residues in proteins under physiological conditions

ABSTRACT

The present invention concerns compounds that block the movement of viral particles through the nuclear membrane and thereby prevent the productive infection of terminally differentiated cells by virus the life cycle of which includes such an importation step. The invention encompasses methods of use of such compounds. The use of the compounds to detect certain specific protein structures which are present in nuclear localization sequences is also taught.

This is a division, of application Ser. No. 08/463,405 filed Jun. 5,1995, which is a continuation-in-part of application Serial No.08/369,830, filed Jan. 6, 1995,now U.S. Pat. No. 5,574,040 which ishereby incorporated by reference in its entirety.

1. FIELD OF THE INVENTION

The field of the present invention concerns compounds that react withspecific sequences in proteins. The present invention more particularlyconcerns a class of compounds that react, under physiclogic conditions,with proteins having adjacent or neighboring lysines. The compounds ofthe invention can be used to label specifically such proteins forresearch purposes and to disrupt their function for pharmacologicpurposes.

2. BACKGROUND TO THE INVENTION 2.1. THE DERIVATIZATION OF PROTEINS

Those skilled in the art will appreciate that there are many compoundsthat can react with specific amino acid residues in proteins, e.g., withsulfhydryl, amino, carboxyl moieties. These reagents are substratespecific, in the sense that each reacts only with one or a few specificamino acids wherever they occur within a protein's sequence. However,the reactivity of such reagents is not affected by the adjacent orneighboring amino acids that form the environment of the reactivemoiety. Thus, the reactivity of such compounds is not context orneighborhood specific.

2.2. NUCLEAR IMPORTATION

The function of an intracellular protein is usually the result of theoverall three dimensional (tertiary) structure of the protein. However,nuclear importation is determined by the simple presence of a shortsequence, called a nuclear localization signal (NLS), which functionsrelatively independently of its position relative to the remainder ofthe structure of object that is imported. In eukaryotic cells allproteins are made in the cytoplasm, which is outside of the nucleus. Ingeneral, those proteins larger than 40 kD that are specificallylocalized in the nucleus of the cell must be actively imported into thenucleus through the nuclear membrane from the cytoplasm via anATP-dependent mechanism that is independent of cell division. Theproteins, and other objects, that are imported have a nuclearlocalization signal (NLS), usually located within the NH₂ terminalsegment of the protein. Several such sequences are known:

a. PKKKRKV from large T antigen of SV40, Kalderon, D., et al., 1984,Cell 39:499-509;

b. AV!KRPAATKKAGQAKKKK LD! from nucleoplasmin, in which only one of thetwo bracketed sequences is required, Dingwall, C., et al., 1988, J. CellBiol. 107:841-49;

c. PRRRRSQS from hepatitis B HbcAg- Yeh, C. T., 1990, J. Virol.

d. KRSAEGGNPPKPLKKLR from the retinoblastoma gene product p110^(rb1)--Zacksenhaus E. et al., 1993, Mol. Cell. Biol. 13:4588

e. KIRLPRGGKKKYKLK from the matrix protein of HIV-1, Bukrinsky, M. I.,et al., 1993, Nature 365:666.

Other viruses that contain NLS sequences include Herpes simplex andmeasles virus. The recognition of an NLS sequence is largely independentof the detailed structure of the object which includes it and of itssite of attachment. Goldfarb, D. S. et al., 1986, Nature 332:641-44;Lanford, R. E., 1986, Cell 46:575. Mere juxtaposition of the amino acidsof the NLS is not sufficient for function, for example NLS function isgenerally not conferred by the peptide having the same sequence of aminoacids in the opposite order as the NLS sequence. Adam, S. A. et al.,1989, Nature 337:276-79.

The primary structure, i.e., the linear sequence, of the NLS mostfrequently contains consecutive lysines, the N.sup.ε moieties of whichpresumably closely approach one another, i.e., they are neighbors.However, certain functional NLS peptides lack consecutive lysines.Robbins, J., et al., 1991, Cell 64:615-23. Presumably the secondary andtertiary structure of these so called "bipartite" NLS peptides givesrise to neighboring N.sup.ε moieties, which may be important for theiractivity.

The cellular proteins or protein complexes that recognize and transportproteins bearing NLS sequences are incompletely understood. It appearsthat there are proteins of the cytoplasmic face of the nuclear membranethat recognize the NLS and, after such recognition, it is this complexthat is transported through the nuclear pore complex. Review: Stochaj,U., et al., 1992, Eur. J. Cell Biol. 59:1-11; Hurt, E. C., 1993, FEBSLetters 325:76-80; Pante, N., et al., 1993, J. Cell. Biol. 122:977-84;Forbes, D. J., 1992, Ann. Rev. Cell Biol. 8:495-527.

A receptor for the NLS sequence has been recently described in a Xenopussystem. Gorlich, D., 1994, Cell 79:767. It is a cytoplasmic 60 kDaprotein which is homologous with previously described proteins ofunknown function, SRP1p of yeast, Yano, R., et al., 1992, Mol. Cell.Biol. 12:5640, and Rch1 of mammals, Cuomo C. A., 1994, Proc. Natl. Acad.Sci. 91:6156.

Two inhibitors of the nuclear localization process have been described.Nuclear localization has been inhibited by lectins (e.g., wheat germagglutinin (WGA)) that bind to the o-linked glycoproteins associatedwith nuclear localization. Dabauvalle, M. -C., 1988, Exp. Cell Res.174:291-96; Sterne-Marr R., et al., 1992, J. Cell Biol. 116:271. Thenuclear localization process, which also depends upon the hydrolysis ofGTP, is blocked by a non-hydrolyzable analog of GTP, e.g., (γ-S) GTP,Melchior, F., 1993, J.Cell Biol. 123:1649.

However, neither (γ-S)GTP nor WGA can be used as pharmaceuticals.Proteins, such as WGA, can be introduced into the interior of a cellonly with considerable difficulty. The same limitation applies tothiotriphospates such as γ-S!GTP. Further, GTPases are involved in amultitude of cell processes and intercellular signaling, thus, the useof a general inhibitor of GTPases would likely lead to unacceptable sideeffects.

2.3. THE SIGNIFICANCE OF NUCLEAR IMPORTATION IN HIV-1 INFECTIONS

Although HIV-1 is a retrovirus, it and other lentiviruses must bedistinguished from viruses of the onco-retrovirus group, which are notassociated with progressive fatal infection. For example, lentivirusesreplicate in non-proliferating cells, e.g., terminally differentiatedmacrophages, Weinberg, J. B., 1991, J. Exp. Med. 172:1477-82, whileonco-retroviruses, do not. Humphries, E. H., & Temin, H. M., 1974, J.Virol. 14:531-46. Secondly, lentiviruses are able to maintain themselvesin a non-integrated, extrachromosomal form in resting T-cells.Stevenson, M., et al., 1990, EMBO J. 9:1551-60; Bukrinsky, M. I., etal., 1991, Science 254:423; Zack, J. L., et al., 1992, J. Virol.66:1717-25. However, it is unclear whether this phenomenon is related tothe presence of latently infected peripheral blood lymphocytes (PBL) inHIV-1 infected subjects, wherein the virus is present in a provirusform. Schnittman, S. M., 1989, Science 245:305; Brinchmann, J. E., etal., 1991, J. Virol. 65:2019; Chapel, A., et al., 1992 J. Virol.66:3966.

The productive infection of a cell by a retroviruses involves the stepsof penetration into the cell, synthesis of a DNA genome from the RNAgenetic material in the virion and insertion of the DNA genome into achromosome of the host, thereby forming a provirus. Both lenti- andoncoretroviruses gain access to the host cell's nucleus during mitosiswhen the nuclear membrane dissolves. However, the lentiviruses are alsoable to cross the nuclear membrane because viral proteins containingnuclear localization sequences are associated with the viralnucleoprotein complex.

The productive infection of terminally differentiated macrophageslocated in the central nervous system is thought to be responsible forthe dementia associated with AIDS. Keonig, S., et al., 1986, Science233:1089; Wiley, C. A. et al., 1986, Proc. Natl. Acad. Sci. 83:7089-93;Price, R. W., et al., 1988, Science 239:586-92. The infection ofterminally differentiated macrophages in the lymphoid system is known tocause aberrant cytokine production. Guilian, D., et al., 1990, Science250:1593; Fauci, A. S., et al., 1991, Ann. Int. Med. 114:678. Thus, thewasting syndrome associated with HIV-1, also known as "slim" disease, isbelieved to be a pathological process that is independent of the loss ofCD4-T-cells. Rather the pathobiology of the wasting is closely relatedto the pathobiology of cachexia in chronic inflammatory and malignantdiseases. Weiss, R. A., 1993, Science 260:1273. For these reasons, theinhibition on HIV-1 infection of macrophages and other non-dividingcells is understood to represent a highly desired modality in thetreatment of HIV-1 infection, especially for patients wherein dementiaor cachexia dominate the clinical picture.

Macrophages play an important role in the transmission of HIV as well.During early stages of the infection, macrophages and cells of themacrophage lineage (i.e. dendritic cells) may be the primary reservoirof HIV-1 in the body, supporting infection of T cells by antigenpresentation activities, Pantaleo, G., et al., 1993, Nature 362:355-358,as well as via the release of free virus. Direct cell-to-celltransmission of the virus may constitute the major route by whichinfection spreads during the early stages of the disease, afterresolution of the initial viremia.

It is noteworthy, in this regard, that macrophage-tropic strains ofHIV-1 predominate in the early stages of infection. Thus, it appearsthat the infection of macrophages is particularly important during thedevelopment of a chronic infective state of the host in a newly infectedsubject. Secondly, macrophages are the HIV-susceptible cell type mostreadily passed during sexual intercourse from an HIV-infected individualinto the circulation of an uninfected individual.

Finally, infection of quiescent T cells by HIV-1 has been shown to takeplace in vitro , Stevenson, M., et al., 1990, EMBO J. 9:1551-1560; Zack,J. A., 1990, Cell 61:213-222, and probably constitutes an importantpathway for the spread of infection in vivo at various stages of thedisease. Bukrinsky, M. I., et al., 1991, Science 254:423-427. AlthoughHIV-1 does not establish productive replication in quiescent T cells,the extrachromosomal retroviral DNA can persist in the cytoplasm of suchcells for a considerable period of time, and initiate replication uponactivation of the host cell. Stevenson, M., et al., 1990, EMBO J.9:1551-1560; Spina, C. A., et al., 1994, J. Exp. Med. 179:115-123;Miller, M.D., et al., 1994, J. Exp. Med. 179:101-113. A recent reportsuggests that the duration of viral persistence in the quiescent T celldepends on the presence of a functional NLS. von Schwedler, U., et al.,1994, Proc. Natl. Acad. Sci. 91:6992-6996. Thus, physicians recognizethe desirability of preventing the infection of macrophages by HIV andunderstand that substantial benefits would be obtained from the use of apharmacologic agent that prevents HIV infection in this cell type.

The mechanism whereby HIV, but not oncoretroviruses, infect non-dividingcells is now understood in broad outline. It is established that thefunction of the pre-integration complex of retrovirus in this regarddoes not depend upon the cellular mechanisms of mitosis or DNAreplication, per se. Rather the integration complex must merely gainaccess to nucleus. Brown, P. O., et al., 1987, Cell 49:347.Onco-retroviruses gain access to the nucleus upon the dissolution of thenuclear membrane in mitosis. By contrast, lentiviruses contain twodistinct proteins that mediate nuclear access through the nuclear porecomplex in the absence of cellular division. For the first of these, thematrix protein (MA or p17), nuclear importation activity is clearly dueto the presence of a trilysyl-containing NLS sequence. Bukrinsky, M. I.,et al., 1993, Nature 365:666; von Schwedler, U., et al., 1994, Proc.Natl. Acad. Sci. 91:6992. A second protein subserving the function ofnuclear entry, the vpr protein, does not contain an identifiable NLSconsensus sequence. Emerman, M., et al., 1994, Nature 369:108;Heinzinger, N. K. et al., 1994, Proc. Natl. Acad. Sci. 91:7311. Rathervpr is thought to form a complex with a cellular protein that doespossess such an NLS sequence.

The significance of the NLS sequence in the importation of HIV-1 intothe nucleus of non-dividing cells has been illustrated in experimentswherein the presence in the medium of a high concentration (0.1 M) ofthe peptide having the sequence of the SV40 T-antigen NLS blocked theimportation of HIV-1 into the nucleus of aphidicolin-arrested CD4⁺ MT4cells. Gulizia, J., et al., 1994, J. Virol. 68:2021-25.

3. SUMMARY OF THE INVENTION

The invention involves a class of aryl alkyl carbonyl compounds,particularly, divalent aryl carbonyl moieties N-linked through the areneto a nitrogen-containing heterocyclic functionality, e.g., an acetyl orpropanoyl substituted aniline moiety N-linked to a pyrimidinium,pyrimidine or triazine moiety. The invention further encompasses methodsof using the compounds of the invention to form tandem Schiff bases inproteins having neighboring N.sup.ε moieties of lysine residues. Asused, herein, neighboring N.sup.ε moieties are two N.sup.ε moieties of aprotein that approach each other as close as the carbonyls of thearylene bis (methyl carbonyl) compounds of the invention, when theprotein is in its natured conformation. As used herein neighboring,adjacent and juxtaposed are equivalent terms in reference to N.sup.εmoieties and refer to the physical locations of the N.sup.ε moieties inthe structure of the native protein and not to the positions of thelysines in the linear sequence.

The invention further encompasses methods of inhibiting productiveinfection by HIV-1 of terminally differentiated (non-dividing cells),particularly macrophages, by inhibition of the importation of thecytoplasmic HIV-1 complex into the nucleus of cell. Particularly theinvention concerns the direct introduction across the cytoplasm membraneof a cell of compounds that block such importation. Thus, in oneembodiment, the invention encompasses methods of using theabove-described compounds to prevent productive infection of terminallydifferentiated macrophages and resting T-cells in HIV-1 infectedsubjects. Without limitation as to theory, the invention is believed toblock the HIV-1 replication by the formation of tandem Schiff bases withneighboring N.sup.ε moieties of vital proteins, a consequence of whichis that the viral nucleoprotein complex does not pass across the nuclearmembrane via interaction with the nuclear pore transport complex and/orother cellular components.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-C. The structures of exemplary Compounds No. 2, 11 and 13 are,respectively, FIGS. 1A, 1B, 1C.

FIG. 2A-C. The effect of various concentrations of Compound No. 2 on RTactivity in the supernatant of HIV-1-infected monocytes. FIG. 2A:Multiplicity of Infection (MOI) 1 ng p24/10⁶ monocytes, cultured inpresence of M-CSF. FIG. 2B: MOI 8 ng p24/10⁶ monocytes, cultured inabsence of M-CSF. FIG. 2C: MOI 0.8 ng p24/10⁶ monocytes, cultured inabsence of M-CSF.

FIG. 3. The effect of various concentrations of Compound No. 2 on RTactivity in the supernatant of HIV-1-infected mitogen-stimulatedperipheral blood leukocytes at infected at 10 and 1.0 ng p24/10⁶ cells,FIG. 3A and 3B, respectively.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. THE COMPOUNDS AND METHODSOF THEIR SYNTHESIS

The compounds of the present invention can be synthesized by reacting adiacetyl or dipropanoyl derivative of aniline with a chloro derivativeof purine, aminomethylpyrimidine, diamino-triazine, or with acyanoguanidine. The reaction can be performed at 90°-100° C. in anaqueous solvent in the presence of a mineral acid to yield thecorresponding aminophenyl pyridine or triazine. The pyrimidinium can besynthesized from the pyrimidine by reaction with an excess methyl iodideat 40°-45° C. under reflux conditions in 1:1acetonitrile/tetrahydrofuran or in a 1:1:2 mixture ofdichloromethane/acetonitrile/tetrahydrofuran.

In a preferred embodiment the compounds of the invention are bis ketonearylene compounds having a third nitrogenous substituent. Thenitrogenous substituent can be further substituted with an aromaticnitrogen-containing heterocyclic compound.

More precisely the compounds of the invention are formed according tothe formula: ##STR1## wherein A═CH₃ or CH₂ CH₃ and ##STR2## whereinX═NH₂, CH₃ or CH₂ CH₃ ; X'═CH₃ or CH₂ CH₃ ; Y═NH₂, NHCH₃, N(CH₃)₂ ; andZ═H, CH₃ or CH₂ CH₃ ; or ##STR3## wherein Y' and Z', independently, ═H,NH₂, NHCH₃, N(CH₃)₂ or N⁺ (CH₃)₃ ; and salts thereof.

5.2. THE INHIBITION OF HIV-1 IMPORTATION INTO THE NUCLEUS OFNON-DIVIDING CELLS

A quantitative measurement of the activity of the compounds of theinvention to block the replication of HIV-1 in non-dividing cells can bedetermined by culture of a macrophage-tropic strain of HIV-1 onperipheral blood-derived macrophages. The cells are cultured for 5-6days prior to infection in a medium consisting of DMEM supplemented with10% type A/B human serum and 200 U/ml Macrophage Colony StimulatingFactor, with half the medium changed after 3 days, to reach a density ofabout 10⁶ cells per 5 ml well. A macrophage-tropic viral stock may begrown on these cells. The concentration of infectious particles in thestock is estimated by measurement of p24 antigen concentration.

To test the effect of compounds of the invention on HIV-1 infection inthe above-described culture system, the medium is removed and replacedwith medium containing HIV-1 at a concentration of 1 ng of p24 (10⁴TCID₅₀ /ml (TCID═tissue culture infectious doses)) and a knownconcentration of the compound of the invention (the inhibitor). After 24hours, the cultures are washed to remove non-adherent virus and theculture is re-fed with medium containing the inhibitor at the desiredconcentration. The amount of replication of HIV-1 is estimated by anassay of the reverse transcriptase activity or by an assay of theconcentration of p24 antigen in the culture medium every 2-3 daysthroughout the post-infection period. In a preferred embodiment theanti-HIV potency of the candidate drug is measured by comparison of theconcentration of reverse transcriptase (RT) or of p24 antigen in themedium of the treated and control cultures at the time of the peak ofthese values in non-treated control cultures, that is about day 5 or 6post-infection. Repetition at various levels of inhibitor allows for thecalculation of the concentration of inhibitor that achieves 50%inhibition of viral growth, IC₅₀. Table I discloses the IC₅₀ of variousinhibitors.

                  TABLE I                                                         ______________________________________                                        Compound                     IC.sub.50                                        ______________________________________                                        2-amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium                                                1 nM                                            iodide (Compound No. 2)                                                       2-amino-4-(3-acetylphenyl)amino-1,6-dimethylpyrimidinium                                                   10 nM                                            iodide (Compound No. 14)                                                      2-amino-4-(3,5-diacetylphenyl)amino-6-methylpyrimidine                                                     50 nM                                            (Compound No. 11)                                                             4-(3-acetylphenyl)amino-2-amino-6-methylpyrimidine                                                         15 nM                                            (Compound No. 15)                                                             ______________________________________                                    

Alternatively, the compounds may all be compared for inhibition of HIVreplication at a fixed concentration. Presented in Table II arecompounds that were used at a concentration of 100 nM to inhibit theproduction of HIV-1 in cultured monocytes infected with HIV-1 10 daysprior to assay (10 ng of p24/10⁶ cells). The production of HIV-1 in eachtreated culture is reported as percentage of untreated control.

                  TABLE II                                                        ______________________________________                                                                    Viral                                             Compound                    Production                                        ______________________________________                                        N-(3,5-diacetylphenyl)biguanide hydrochloride (Compound                                                   12%                                               No. 12)                                                                       2-(3,5-diacetylphenyl)amino-4,6-diamino-1,3,5-triazine                                                    14%                                               (Compound No. 13)                                                             4-(3-acetylphenyl)amino-2-amino-6-methylpyrimidine                                                        20%                                               (Compound No. 17)                                                             3,5-diacetylaniline         20%                                               N,N-dimethyl-3,5-diacetylaniline                                                                          25%                                               2,6-diacetylaniline         28%                                               3,5-diacetylpyridine        58%                                               ______________________________________                                    

FIG. 2A presents further results of the use of the most active of thecompounds of Table I, Compound No. 2, to block the replication of HIV-1in purified monocytes, cultured in medium supplemented withmonocyte-colony stimulating factor (M-CSF). The cultures were treatedwith none or between 10⁻¹² and 10⁻⁶ M Compound No. 2 and, simultaneouslywith the beginning of treatment, the cells were exposed to themonocyte-tropic strain HIV-1_(ADA) at about 0.01 TCID₅₀ /cell (1 ngp24/10⁶ cells) for 2 hours. Samples were withdrawn at days 3, 6, 10, 14and 17 after infection and assayed for reverse transcription activity.Compound No. 2 does not inhibit reverse transcriptase, data not shown.The results show that under these conditions the IC₅₀ concentrations isbetween 0.1 and 1.0 nM and that a concentration of between 0.1 μM and1.0 μM completely inhibits the replication of the virus.

FIGS. 2B and 2C show the effects of various concentrations of CompoundNo. 2 on the production of HIV-1 in monocyte cultures not supplementedwith M-CSF. In these studies MOI, as determined by concentration of p24antigen was; FIG. 2B (8 ng/10⁶ cells) and FIG. 2C (0.8 ng/10⁶ cells).These experiments showed IC₅₀ s of about 10 nM and of less than 1.0 nMrespectively.

The inhibition of the replication of HIV-1 is not due to generalcytotoxic effects of the compound. Concentrations of Compound No. 2 ashigh as 10 μM were without toxic effects on the monocyte cultures asdetermined by lactate dehydrogenase release and trypan blue exclusion.Further evidence of the specificity of the inhibition due to CompoundNo. 2 is provided by the data presented in FIG. 3A and 3B whereinmitogen-stimulated peripheral blood leukocytes were cultured inIL-2-supplemented medium and were exposed to the HIV-1_(ADA) at p24concentrations of 10 and 1 ng/10⁶ cells, respectively. In thisexperiment up to 10 μM Compound No. 2 had only a marginal effect onvital production at the higher MOI. At the lower MOI, 1 and 10 μM ofCompound No. 2 caused an approximate 2-fold reduction in viral output.

The inhibition of HIV-1 importation into the nucleus of non-dividingcells can also be directly measured. One suitable method to determinedirectly the activity of compounds of the invention utilizes a cell linethat is susceptible to HIV-1 infection, e.g., MT-4 cells, that is growtharrested by treatment with aphidicolin and exposed to HIV-1. PCRamplification is used to detect double-stranded closed circular HIV-1genomes, which are formed only after nuclear importation, by selectingprimers that bridge the junction point of the genome. For greater detailsee Bukrinsky, M. I., et al., 1992, Proc. Natl. Acad. Sci. 9:6580-84.

5.3. THE TREATMENT OF HIV INFECTION

The present invention provides a method of treatment of HIV-1 infectionby administering to an HIV-1-infected subject a pharmaceuticalcomposition having, as an active ingredient, an effective amount of acompound of the invention. In one embodiment the compound to beadministered is Compound No. 2. Pharmaceutical compositions suitable fororal, intraperitoneal, and intravenous administration can be used in thepractice of the invention. Such pharmaceutical compositions include, byway of non-limiting examples, aqueous solutions of the chloride,bicarbonate, phosphate and acetate salts of Compound No. 2 andpH-buffered mixtures thereof.

The effective dose of the active ingredient can be determined by methodswell known to those skilled in medicinal chemistry and pharmacology. Aneffective dose is the dose that achieves in the subject's plasma aconcentration of the active ingredient that is sufficient to inhibit thereplication of HIV-1 in monocyte cultures as described in Section 5.4,supra, but does not lead to cytopathic effects in such cultures.

The daily dose and dosing schedule to be given a subject can bedetermined by those skilled in the art, using the pharmacokineticconstants set forth in Table III below, to achieve a target plasmaconcentration. The target plasma concentration can be selected byroutine pharmacological and clinical investigation methods well-known tothose skilled in the art, and can be based on a range of concentrationswhich encompass the IC₅₀ calculated for each particular compound. Forexample, the dose can be adjusted to achieve a range of target plasmaconcentrations that included the IC₅₀ for the compounds as shown inTable I above.

                  TABLE III                                                       ______________________________________                                        Pharmacokinetic constants                                                     Compound             No. 2   No. 11  No. 15                                   ______________________________________                                        Area under the plasma                                                                      AUC    μg*hr/ml                                                                            16.84 3.93  0.817                                curve                                                                         Distribution rate contant                                                                  α                                                                              hr.sup.-1                                                                              1.1193                                                                              1.8286                                                                              2.14                                 Elimination rate constant                                                                  β hr.sup.-1                                                                              0.0046                                                                              0.1865                                                                              0.0398                               Initial distribution plasma                                                                A      μg/ml 13.996                                                                              5.22  1.10                                 level                                                                         Initial elimination plasma                                                                 B      μg/ml 0.054 0.14  0.0082                               level                                                                         Distribution phase half-                                                                   t.sub.1/2 α                                                                    hr       0.62  0.38  0.32                                 life                                                                          Elimination phase half-                                                                    t.sub.1/2 β                                                                     hr       150.46                                                                              3.72  17.41                                life                                                                          Volume of distribution                                                                     V.sub.β                                                                         liters   18.33 7.07  48.29                                Total clearance rate                                                                       Cl.sub.S                                                                             ml/min   1.4   22    32                                   ______________________________________                                    

For example, using the foregoing pharmacokinetic constants,particularly, the clearance rate, the daily dose and dosing scheuleneeded to obtain a given target average plasma concentration can becalculated. The results of such calculations for Compound Nos. 2, 11 and15 are presented in Table IV. The calculated doses of Compound Nos. 2and 15 are considerably below the toxic levels, as measured by the LD₅₀,of these compounds. See, Section 6.4 below.

                  TABLE IV                                                        ______________________________________                                        Compound        Target     Clearance‡                                                                  Dose                                      No.     M.W.    serum conc.                                                                              (ml/min) (mg/Kg day)                               ______________________________________                                         2*     334     10 nM      1.4      0.266                                     11      280     50 nM      22       17.5                                      15      250     15 nM      32       6.7                                       ______________________________________                                         ‡measured in a 25 gr mouse                                         *Chloride salt                                                           

Using such methods, a dose can be calculated to achieve a predeterminedtarget plasma concentration. A practicable target plasma concentrationof Compound No. 2 ranges from 0.5 nM to 10 nM; for Compound No. 11, apracticable target range is from 25 nM to 100 nM; for Compound No. 15, apracticable target range is from 7.5 nM to 50 nM.

Subjects who can benefit from the administration of the compounds of theinvention according to this method include all persons infected byHIV-1. More particularly, firstly, those who benefit include thosesubjects who have or are at risk to develop CNS signs of HIV-1 infectionand/or subjects that have developed significant weight loss. Secondly,those who benefit include those who have been recently exposed to HIV-1,but who do not yet have an established chronic infection.

5.4. PHARMACEUTICAL FORMULATIONS

Because of their pharmacological properties, the compounds of thepresent invention can be used especially as agents to treat patientssuffering from HIV and can be used as agents to treat patients sufferingfrom other viral infections or chronic diseases that are dependent uponnuclear localization as part of the pathogenic process. Such a compoundcan be administered to a patient either by itself, or in pharmaceuticalcompositions where it is mixed with suitable carriers or excipient(s).

Use of pharmaceutically acceptable carriers to formulate the compoundsherein disclosed for the practice of the invention into dosages suitablefor systemic administration is within the scope of the invention. Withproper choice of carrier and suitable manufacturing practice, thecompositions of the present invention, in particular, those formulatedas solutions, may be administered parenterally, such as by intravenousinjection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well-known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levitating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

5.5. USE OF THE COMPOUNDS OF THE INVENTION TO DERIVATIZE PROTEINS

The compounds of the present invention can be used to derivatize atarget protein and thereby determine the presence of adjacent N.sup.ε-moieties. The test reaction can be conducted in aqueous buffer at mildto moderate alkaline pH, between about 7.2 and 8.0. Specificderivatization of the target protein can be detected by any means thatseparates protein-bound and free derivatizing compound. The derivatizingcompound optionally can be detected by radiolabeling it. In oneembodiment, the compound can be synthesized using ¹⁴ C-methyliodide inplace of methyliodide. Alternatively, use can be made of the strong UVabsorption or fluorescence of the derivatizing compounds. Compound No.2, for example has a absorption peak of 16,000 M⁻¹ cm⁻¹ at λ=298 nm. Ina preferred embodiment the target protein is derivatized by a compoundof the invention, irreversibly reduced with sodium borohydride orcyanoborohydride and fragmented into peptides by trypsin or the like.The resultant peptides can be compared with the peptides obtained froman unreacted sample of the protein by analysis using any chromatographicor electrophoretic technique that resolves peptides, e.g., reverse phaseHigh Performance Liquid Chromatography (HPLC). When the peptides areresolved by any high resolution chromatography procedure, thederivatized peptides can be readily detected by their altered elutiontime and the absorbance at λ=298 nm.

In a preferred embodiment the practitioner will conduct the reaction atvarious pH points to determine whether a positive result can be obtainedat any point within the expected range. A positive result, i.e., aresult that indicates the presence of adjacent N.sup.ε -moieties, is onein which a large fraction of each of a limited number, i.e., between1-4, of peptides of the target protein are derivatized and negligibleamounts of other peptides are affected.

The above-described protein derivatization technique can be used todetermine whether a candidate compound can be used, according to theinvention to prevent productive HIV-1 infection of macrophages. Acomparison of the activity of a candidate compound and that of CompoundNo. 2 as derivatizing agents specific for nuclear localization sequencescan be made. A compound that derivatizes the same peptides to the sameextent as Compound No. 2 can be used to practice the invention.

6. EXAMPLES 6.1. SYNTHESIS OF SPECIFIC COMPOUNDS

Compound No. 2, FIG. 1A: A suspension of Compound No. 11(2-amino-4-(3,5-diacetylphenyl)amino-6-methylpyrimidine) (0.284 g), wassuspended in 1:1 acetonitrile-tetrahydrofuran was treated with methyliodide (2 mL) and heated at 40°-45° C. under a reflux condenser for 18hr. Cooling and filtration gave 0.35 g of2-amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium iodide, mp292° C.

Compound No. 11: A suspension of 3,5-diacetylaniline (0.885 g) in water(18 mL) was treated with 2-amino-4-chloro-6-methylpyrimidine (0.718 g)and concentrated HCl (0.42 mL) and heated at 90°-100° C. for 30 min.After cooling the mixture was treated with 10 mL of aqueous 1N KOH. Themixture was stirred for 10 min and the solid was filtered out, washedwith water, and dried, to give 1.332 g of tan crystals.Recrystallization from ethyl acetate-2-methoxyethanol gave 1.175 g of2-amino-4-(3,5-diacetylphenyl)amino-6-methylpyrimidine as light buffcrystals, mp 240°-241° C.

Compound No. 12. A suspension of 3,5-diacetylaniline (0.531 g) in water(8 mL) was treated with cyanoguanidine (0.285 g) and conc. HCl (0.25 mL)and heated at reflux. After 6 hr the mixture was cooled and concentratedand 0.248 g of off-white solid was filtered out and dried to giveN-(3,5-diacetylphenyl)biguanide hydrochloride, mp 260°-70° C. (dec).

Compound No. 13: A suspension of 3,5-diacetylaniline (1.95 g) in water(10 mL) was treated with 2-chloro-4,6-diamino-1,3,5-triazine (1.455 g)and concentrated HCl (0.1 mL) and heated at reflux for 20 min. Aftercooling the hydrochloride of Compound No. 13 separated as a whitepowder. This was filtered out, dissolved in 60 mL of boiling aqueous 75%methanol and treated with triethylamine (1.5 mL). On cooling, off-whiteflakes separated. Filtration and drying gave 1.79 g of2-(3,5-diacetylphenyl)amino-4,6-diamino-1,3,5-triazine, mp 271°-2° C.

Compound No. 14: 4-(3-acetylphenyl)amino-2-amino-6-methylpyrimidine,Compound No. 15, (0.968 g) was suspended in acetone (5 mL) containingmethyl iodide (2 mL) was heated at reflux for 48 hr. Filtration aftercooling gave 0.657 g of4-(3-acetylphenyl)amino-2-amino-1,6-dimethylpyrimidinium iodide as awhite powder, mp 238°-40° C.

Compound No. 15: A suspension of m-aminoacetophenone (2.7 g) and2-amino-4-chloro-6-methylpyrimidine (2.87 g) in 40 mL water was treatedwith 1.7 mL concentrated HCl and heated at reflux for 1 hour. Additionof 40 mL 1N KOH gave a light buff solid, which was filtered out anddried to give 3.8 g 4-(3-acetylphenyl)amino-2-amino-6-methylpyrimidine,mp 196°-98° C.

Compound No. 16: A suspension of 3,5-diacetylaniline (0.531 g) in water(10 mL) was treated with 6-chloropurine (0.464 g) and concentrated HCl(0.25 mL) and heated at reflux for 30 min. After cooling the mixture wastreated with 6 mL of aqueous 1N KOH. The mixture was stirred for 10 minand the solid was filtered out, washed with water, and dried, to give0.80 g of 6- (3,5-diacetylphenyl)amino! purine, mp dec 340°-350° C.

Compound No. 17: A suspension of p-aminoacetophenone (1.35 g) and2-amino-4-chloro-6-methylpyrimidine (1.435 g) in 20 mL water was treatedwith 0.85 mL conc HCl and heated at reflux for 1 hr. Addition of 20 mL1N KOH gave a light buff solid, which was filtered out and dried to give2.28 g 4-(3-acetylphenyl) amino-2-amino-6-methylpyrimidine, mp 194°-196°C. Of this, 1.21 g was treated with methyl iodide (3 mL) indimethylformamide (15 mL) at room temperature for 42 hr. Dilution withethyl acetate and filtration gave 1.11 g 4-(4-acetylphenyl)amino-2-amino-1,6-dimethylpyrimidinium iodide as a white powder, mp302°-3° C.

6.2. THE USE OF COMPOUND NO. 2 TO INHIBIT HIV REPLICATION IN PRIMARYMACROPHAGE LINES. 6.2.1. Materials and Methods.

Primary human monocytes were obtained from peripheral blood byFicoll-Hypaque centrifugation and adherence to plastic as describedpreviously. Gartner S. P., et al., 1986, Science 233:215. Briefly, afterFicoll-Hypaque (Pharmacia) separation, PBMCs were washed 4 times withDMEM (the last wash was done at 800 rpm to remove platelets) andresuspended in monocyte culture medium DMEM supplemented with 1 mMglutamine, 10% heat-inactivated human serum, 1% penicillin+streptomycinmixture (Sigma)! at a density of 6×10⁶ cells/ml. Cells were seeded in24-well plates (1 ml per well) and incubated for 2 h at 37° C., 5% CO₂.Following incubation, cells were washed 3 times with DMEM to removenon-adherent cells and incubation was continued in monocyte culturemedium supplemented with 250 U/ml human M-CSF (Sigma). Cells wereallowed to mature for 7 days prior to infection with the monocyte-tropicstrain, HIV-1_(ADA). Nuovo, G. J., et al., 1992, Diagn. Mol. Pathol.1:98. Two hours after infection, cells were washed with medium andcultured in RPMI supplemented with 10% human serum. In experiments wherePCR analysis was performed, virus was pretreated with RNAse-free DNAse(Boehinger-Mannheim) for 2 h at room temperature and then filteredthough a 0.2 μm pore nitrocellulose filter prior to infection.

PBMCs were purified by Ficoll-Hypaque centrifugation and activated by 10μg/ml PHA-P (Sigma) and 20 U/ml recombinant human IL-2 (rhIL-2) in RPMI1640 supplemented with 10% FBS (HyClone). After 24 h incubation, cellswere washed and inoculated with HIV-1_(ADA) in RPMI 1640 supplementedwith 10% FBS. After a 2 h adsorption, free virus was washed away andcells were cultured in RPMI 1640 supplemented with 10% FBS and 20 U/mlrhIL-2.

Virus stock and infection. Macrophage-tropic strain HIV-1_(ADA) wasamplified in primary human monocytes and concentrated to produce stockwith TCID₅₀ of about 10⁵ /ml. The concentration of HIV-1 was determinedby immunoassay of viral p24, concentration; using a conversion factor of1 ng/200 HIV-1 particles.

6.2.2. p24 and RT Assay

For p24 assay, sequential 1:9 dilutions of culture supernatant wereprepared and analyzed by ELISA as suggested by the manufacturer(Cellular Products, Buffalo, N.Y.). For the reverse transcriptase (RT)assay, 10 μl of culture supernatant was added to 40 μl of reactionmixture (final composition was 50 mM Tris-HCl, pH 7.8; 20 mM KCl; 5 mMMgCl₂ ; 1 mMDTT; 0.1% Triton X-100; 0.2 OD/ml polyA; 0.2 OD/mloligo(dT)₁₂₋₁₈ ; and 40 μCi/ml ³ H-dTTP (76 Ci/mmol, DuPont) andincubated 2 hr at 37° C. 5 μl of the reaction mixture was then spottedonto the DE 81 (Whatman) paper. Paper was air dried and washed 5 timeswith 5% Na₂ HPO₄, followed by rinsing with distilled water. After airdrying, paper was put on a Flexi Filter plate (Packard), covered withscintillation fluid and counted in a Top Count Microplate Counter(Packard). Results are expressed as counts per minute in 1 ml ofsupernatant (cpm/ml).

6.2.3. Results Dividing and Quiescent Cells

The cytotoxicity of Compound No. 2 was tested in monocyte cultures bytrypan blue exclusion assay or lactate dehydrogenase (LDH) release. Byboth assays, no cytotoxic effect was observed with concentrations of thecompound up to 10 μM (data not shown). Results presented in FIG. 2 showthe effect of various concentrations of Compound 2 on HIV-1 replicationin monocytes. From this experiment, we estimate the IC₅₀ for thiscompound between 0.1 and 1 nM. Similar and higher concentrations of thecompound were also tested on activated PBLs. The anti-vital effect ofthis compound was much less expressed in these actively dividing cellpopulations (FIG. 3). No anti-viral effect was detected when cultures ofreplicating cells were infected at the multiplicity of infection used toinfect monocytes.

6.2.4. AZT and Compound No. 2 in Combination

AZT is a drug that is routinely used to treat HIV-1 infected persons.However, two factors are known to diminish the effectiveness of AZT: itstoxicity and the emergence of resistant mutant strains of HIV-1. Theeffects of both of these factors can be reduced by administering asecond, synergistic HIV-1-inhibitory drug with AZT.

In view of these premises, the effects on HIV-1 replication in humanmonocyte cultures of the various concentrations of AZT, alone or or incombination with 100 nM Compound No. 2, were tested using the protocolsof Sections 6.2.1 and 6.2.2. Drugs were added to the monocyte culturestogether with HIV-1 at about 10⁵ TCID/ml. The concentration of drugs wasmaintained on refeeding. HIV-1 replication was assessed by assay of thesupernatant for reverse transcriptase activity. The results areexpressed as mean±std. der. (cpmx10⁻³) in Table V.

                  TABLE V                                                         ______________________________________                                        Effects of Combined AZT/Compound No. 2                                        on HIV-1 infected Monocyte Cultures                                                  day-7         day-11                                                    AZT!    (-) No. 2 (+) No. 2 (-) No. 2                                                                             (+) No. 2                                ______________________________________                                        0        1.46 ± 0.43                                                                          0.37 ± 0.07                                                                          1.81 ± 0.75                                                                        0.72 ± 0.30                           10    pM     0.92 ± 0.21                                                                          0.15 ± 0.05                                                                        1.63 ± 0.81                                                                        0.18 ± 0.06                         100   pM     0.79 ± 0.14                                                                          0.13 ± 0.04                                                                        1.34 ± 0.59                                                                        0.15 ± 0.06                         1     nM     0.60 ± 0.28                                                                          0.04 ± 0.02                                                                        1.07 ± 0.49                                                                        0.09 ± 0.03                         10    nM     0.05 ± 0.02                                                                          0.03 ± 0.02                                                                        0.08 ± 0.03                                                                        0.07 ± 0.03                         ______________________________________                                    

These results demonstrate that there is synergy between the AZT andCompound No. 2. The synergistic effects are most pronounced at the lowerdoses of AZT on day 11. For example, 10 pM AZT alone produces an about20% reduction in RT activity on day-11, 100 nM Compound No. 2 aloneproduces about a 60% reduction. Without synergy, the combination shouldproduce a 70% reduction (100×(1-(0.8×0.4)). Instead the observedreduction was 90%.

6.3. THE COMPOUNDS OF THE INVENTION DO NOT BLOCK THE NUCLEAR IMPORTATIONOF ESSENTIAL PROTEINS IN CELLS 6.3.1. Direct Demonstration of theInhibition of HIV-1 Nuclear Importation by Compound No. 2

The effects of Compound No. 2 on the nuclear importation of HIV-1preintegration complexes can be directly measured by detecting thepresence of circularized duplex HIV-1 genomic DNA. These duplex circlescan be readily detected by PCR amplification using primers which spanthe junction of the circularized HIV-1 genome. Bukrinsky, M. I., et al.,1992, Proc. Natl. Acad. Sci. 89:6580-84.

Briefly, the efficiency of nuclear translocation was estimated by theratio between the 2-LTR- and pol -specific PCR products, which reflectsthe portion of 2-LTR circle DNA molecules as a fraction of the entirepool of intracellular HIV-1 DNA. Viral 2-LTR circle DNA is formedexclusively within the nucleus of infected cells and thus is aconvenient marker of successful nuclear translocation. Bukrinsky, M. I.,1992, Procd. Natl. Acad. Sci. 89:6580-84; Bukrinsky, M. I., 1993, Nature365:666-669.

PCR analysis of HIV-1 DNA: Total DNA was extracted from HIV-1-infectedcells using the IsoQuick extraction kit (Microprobe Corp., Garden Grove,Calif.). DNA was then analyzed by PCR using primer pairs that amplifythe following sequences: a fragment of HIV-1 (LTR/gag) that is the lastone to be synthesized during reverse transcription and thereforerepresents the pool of full-length viral DNA molecules; a fragment ofpolymerase gene (pol); a 2-LTR junction region found only in HIV-1 2-LTRcircle DNA molecules; or a fragment of the cellular a-tubulin gene.Dilutions of 8E5 cells (containing 1 integrated copy of HIV-1 DNA pergenome) into CEM cells were used as standards. Amplification productswere transferred to nylon membrane filters and hybridized to ³²P-labeled oligonucleotides corresponding to internal sequences specificfor each PCR amplification fragment, followed by exposure to Kodak XAR-5film or a phosphor screen.

Quantitation of PCR Reactions: Bands of correct size revealed afterhybridization were quantitated with a PhosphorImager (MolecularDynamics) by measuring the total density (integrated volume) ofrectangles enclosing the corresponding product band. Efficiency ofnuclear translocation of HIV-1 DNA was estimated by measurement of theamount of 2-LTR circle DNA (N_(2-LTR)) relative to total viral DNA(N_(tot)) in each culture, indexed to the same ratio of appropriatecontrol cultures. Thus, Translocation Index=(N_(2-LTR)/N_(tot))/(C_(2-LTR) / C_(tot))×100.

Results: Primary human monocytes were infected with HIV-1_(ADA) in thepresence of 100 nM concentration of Compound No. 2 or without drugs(control). Half the medium was changed every 3 days, and drugs werepresent throughout the whole experiment. Cell samples were taken at 48and 96 hours post infection and the Translocation Index, relative to thedrug free control was determined. At both time points the TranslocationIndex was less than 10, indicating there was greater than 90% inhibitionof nuclear importation.

6.4. PHARMACOKINETIC AND TOXICOLOGICAL STUDIES

Toxicity: Acute whole animal toxicity determinations were made forseveral compounds of the present invention by estimating LD₅₀ valuesaccording to standard protocols. Compound 2 was emulsified in 10%DMSO/peanut oil; Compound 15 was dissolved in dH₂ O titrated with HCl.Test compounds were administered by i.p. injection to outbred SwissWebster ND4 mice (five mice per dosage group) according to adose-doubling protocol beginning at 2.5 mg/kg body weight (i.e. doses of2.5, 5, 10, 20, 40, 80, 160, 320, 640 mg/kg etc.) as required toestablished the LD₅₀. Mice were observed for 3-7 days followingtreatment, which comprised a single bolus injection; deaths and otherovert signs of toxicity were recorded. LD₅₀ values were calculated fromthe data by non-linear regression analysis using the Chou equation,(Chou, 1976, J. Theor. Biol. 39: 253-276), and are shown in the tablebelow:

    ______________________________________                                        Acute toxicity                                                                            LD.sub.50 in mg/kg (mean ± s.e.m.)                             ______________________________________                                        Compound No. 2                                                                              587.77 ± 65.76                                               Compound No. 15                                                                             49.04 ± 0.08                                                 ______________________________________                                    

Pharmacokinetics: Some basic pharmacokinetic constants were estimatedfrom the appearance and clearance of the compounds of the presentinvention in the blood of outbred Swiss Webster ND4 mice given singlei.p. doses of compound, according to standard protocols well known inthe art. Compound 2 was emulsified in 10% DMSO/peanut oil andadministered at 50 mg/kg. Compound 11 was dissolved in water titratedwith HCl and administered at 50 mg/kg. Compound 15 was dissolved inwater titrated with HCl and administered at 20 mg/kg. Blood samples weretaken at intervals after bolus drug delivery, and the levels of compoundwere determined by ion-pair, reverse-phase chromatography utilizing aCH₃ CN gradient in 10 mM heptane sulfonate/10 mM tetramethylammoniumchloride/4.2 mM H₃ PO₄ (Buffer A) and a Zorbax RX-C8 column. Columneluate was monitored at 300 nm with 500 nm as a reference wavelength.The analyte was extracted and concentrated by passing a known volume ofserum over a cyanopropyl solid-phase extraction column and eluting with95% CH₃ CN/5% H₂ O containing 10 mM heptane sulfonate/10 mMtetramethylammonium chloride/4.2 mM H₃ PO₄. Eluted samples wereevaporated to dryness and redissolved in Buffer A. Approximate valuesfor pharmacokinetic constants were calculated from the serumconcentrations using the method of residuals. See, Table III of Section5.3 above.

Oral Availability

Compound No. 2 was dissolved in dH₂ O and given to outbred mice at adose of 50 mg/kg by garage. Bioavailability was estimated at 18%relative to i.p. injection (above) by monitoring plasma appearance andclearance in blood samples taken at intervals after this single bolusoral treatment. Maximal plasma concentrations (C_(max)) occurred atabout 60 min, reaching a level of about 0.41 μg/ml.

The present invention is not to be limited in scope by the specificembodiments described which were intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components were within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

We claim:
 1. A pharmaceutical composition comprising a compoundaccording to the formula: ##STR4## wherein A═CH₃ or CH₂ CH₃ and ##STR5##wherein X═NH₂, CH₃ or CH₂ CH₃ ; X'═CH₃ or CH₂ CH₃ ; Y═NH₂, NHCH₃,N(CH₃)₂ ; and Z═H, CH₃ or CH₂ CH₃ ; or ##STR6## wherein Y' and Z',independently,═H, NH₂, NHCH₃, N(CH₃)₂ or N⁺ (CH₃)₃ or a salt thereof; atherapeutically effective amount of AZT; and a pharmaceuticallyacceptable carrier.
 2. The pharmaceutical composition of claim 1comprising compound according to the formula: ##STR7## or a saltthereof, wherein A═CH₃, X═NH₂, CH₃ or CH₂ CH₃ ; X'═CH₃ or CH₂ CH₃ ;Y═NH₂, NHCH₃, N(CH₃)₂ ; and Z═H, CH₃, or CH₂ CH₃.
 3. The pharmaceuticalcomposition of claim 2 wherein Z═H, Y═NH₂, X and X'═CH₃ and saltsthereof.
 4. A method of treatment of HIV-1 infection comprisingadministering to an HIV-1-infected subject the pharmaceuticalcomposition of claim
 3. 5. A method of treatment of HIV-1 infectioncomprising administering to an HIV-1-infected subject the pharmaceuticalcomposition of claim
 1. 6. A method of treatment of HIV-1 infectioncomprising administering to an HIV-1-infected subject the pharmaceuticalcomposition of claim 2.